Use of a gene for increasing the oil content in plants

ABSTRACT

The invention relates to methods for increasing the oil content in plants, preferably in plant seeds, by expressing a polypeptide from yeast. The invention furthermore relates to expression constructs for expressing the yeast polypeptide in plants, preferably in plant seeds, the transgenic plants expressing the yeast polypeptide and to the use of said transgenic plants for the production of food, feeds, seed, pharmaceuticals or fine chemicals, in particular for the production of oils.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371)of PCT/EP2003/007084 filed Jul. 3, 2003, which claims benefit ofEuropean application 02015344.1 filed Jul. 10, 2002.

The invention relates to the use of a gene that when expressed willincrease the total amount of oil (i.e. triacylglycerols—TAG) that isproduced in transgenic organisms.

More specifically this invention describes the identification of a geneencoding a TAG synthesis enhancing protein (TEP).

In a first embodiment, this invention is directed to the TEP proteincomprising an amino acid sequence as set forth in SEQ ID NO: 2 or afunctional fragment, derivative, variant, or ortologue thereof.

The present invention further includes the nucleotide sequence as setforth in SEQ ID NO: 1, as well as portions of the genomic sequence, thecDNA sequence, allelic variants, synthetic variants and mutants thereof.This includes sequences that are to be used as probes, vectors fortransformation or cloning intermediates.

SEQ ID NO. 2 is the deduced amino acid sequence from the open readingframe YJR098c in SEQ ID NO. 1.

Another aspect of the present invention relates to those polypeptides,which have at least 60% identity to SEQ ID NO: 2.

The invention furthermore relates to expression constructs forexpressing yeast TEP in plants, preferably in plant seeds, transgenicplants expressing yeast TEP, and to the use of said transgenic plantsfor the production of food, feeds, seed, pharmaceuticals or finechemical, in particular for the production of oils.

In oil crops like rape, sunflower, oil palm etc., the oil (i.e.triacylglycerols) is the most valuable product of the seeds or fruitsand other compounds such as starch, protein and fiber is regarded asby-products with less value. Enhancing the quantity of oil per weightbasis at the expense of other compounds in oil crops would thereforeincrease the value of the crop. If proteins that promote the allocationof reduced carbon into the production of oil can be up regulated byoverexpression, the cells will accumulate more oil at the expense ofother products. This approach could not only be used to increase the oilcontent in already high oil producing organisms such as oil crops, theycould also lead to significant oil production in moderate or low oilcontaining crops such as soy, oat, maize, potato, sugar beats, andturnips as well as in microorganisms.

Increasing the oil content in plants and, in particular, in plant seedsis of great interest for traditional and modern plant breeding and inparticular for plant biotechnology. Owing to the increasing consumptionof vegetable oils for nutrition or industrial applications,possibilities of increasing or modifying vegetable oils are increasinglythe subject of current research (for example Töpfer et al. (1995)Science 268:681-686). Its aim is in particular increasing the fatty acidcontent in seed oils.

The fatty acids which can be obtained from the vegetable oils are alsoof particular interest. They are employed, for example, as bases forplasticizers, lubricants, surfactants, cosmetics and the like and areemployed as valuable bases in the food and feed industries. Thus, forexample, it is of particular interest to provide rapeseed oils withfatty acids with medium chain length since these are in demand inparticular in the production of surfactants.

The targeted modulation of plant metabolic pathways by recombinantmethods allows the modification of the plant metabolism in anadvantageous manner which, when using traditional breeding methods,could only be achieved after a complicated procedure or not at all.Thus, unusual fatty acids, for example specific poly-unsaturated fattyacids, are only synthesized in certain plants or not at all in plantsand can therefore only be produced by expressing the relevant gene intransgenic plants (for example Millar et al. (2000) Trends Plant Sci5:95-101).

Triacylgylcerides and other lipids are synthesized from fatty acids.Fatty acid biosynthesis and triacylglyceride biosynthesis can beconsidered as separate biosynthetic pathways owing to thecompartmentalization, but as a single biosynthetic pathway in view ofthe end product. Lipid synthesis can be divided into twopart-mechanisms, one which might be termed “prokaryotic” and anotherwhich may be termed “eukaryotic” (Browse et al. (1986) Biochemical J235:25-31; Ohlrogge & Browse (1995) Plant Cell 7:957-970). Theprokaryotic mechanism is localized in the plastids and encompasses thebiosynthesis of the free fatty acids which are exported into thecytosol, where they enter the eukaryotic mechanism in the form of fattyacid acyl-CoA esters and are esterified with glycerol-3-phosphate (G3P)to give phosphatidic acid (PA). PA is the starting point for thesynthesis of neutral and polar lipids. The neutral lipids aresynthesized on the endoplasmic reticulum via the Kennedy pathway(Voelker (1996) Genetic Engineering, Setlow (ed.) 18:111-113; Shankline& Cahoon (1998) Annu Rev Plant Physiol Plant Mol Biol 49:611-649;Frentzen (1998) Lipids 100:161-166).

The last step in the synthesis of triacylglycerols has been shown tooccur by two different enzymatic reactions, an acyl-CoA dependentreaction catalyzed by an acyl-CoA:diacylglycerol acyltransferase (Cases,et al., 1998; Lardizabal, et al., 2001) and the acyl-CoA independentreaction catalyzed by an phospholipid:diacylglyerol acyltransferase(Dahlqvist, et al., 2000). Two unrelated gene families encodingacyl-CoA:diacylglycerol acyltransferases have been identified in plants,animals and yeast, whereas the gene family encoding the acyl-CoAindependent enzyme has been identified in yeast but not in plants oranimals. In yeast, a total of four genes (are1, are2, lro1, dga1) belongto these three gene families, and they are the only genes known tocontribute directly to triacylglycerol synthesis. Thus, no synthesis oftriacylglycerol could be detected in yeast cells where all four geneswere disrupted. In the present invention we show, that a fifth gene ispresent in yeast, which enhances the amount of triacylglycerol thataccumulates in wildtype yeast.

It is an object of the present invention to provide alternative methodsfor increasing the oil content in plants.

We have found that this object is achieved by the present invention.

A first subject matter of the invention comprises a method of increasingthe total oil content in a plant organism or a tissue, organ, part, cellor propagation material thereof, comprising

-   a) the transgenic expression of yeast TEP in said plant organism or    in a tissue, organ, part, cell or propagation material thereof, and-   b) the selection of plant organisms in which—in contrast to or    comparison with the starting organism—the total oil content in said    plant organism or in a tissue, organ, part, cell or propagation    material thereof is increased.

Other proteins resulting in the same effect as the protein set forth inSEQ ID NO. 2 are obtainable from the specific sequences provided herein.Furthermore, it will be apparent that one can obtain natural andsynthetic TEPs, including those with modified amino acid sequences andstarting materials for synthetic-protein modeling from the exemplifiedTEPs and from TEPs which are obtained through the use of suchexemplified sequences. Modified amino acid sequences include sequencesthat have been mutated, truncated, increased and the like, whether suchsequences were partially or wholly synthesized.

Further, the nucleic acid probes (DNA or RNA) derived from the SEQ-IDNo. 1 of the present invention can be used to screen and recover“homologous” or “related” sequences from a variety of plant andmicrobial sources.

The present invention can be essentially characterized by the followingaspects:

Example 1 shows the reduction of triacylglycerol accumulation in yeastcells lacking the YJR098c gene.

Example 2 shows the increased accumulation of triacylglycerol in yeastcells expressing the YJR098c gene in combination with a strong promoter.

Example 3 shows a significantly higher total oil content in the seeds oftransgenic plant lines with increased expression of the YJR098c geneconstruct.

Use of a nucleic acid sequence SEQ-ID No: 1, encoding a protein SEQ-IDNo: 2 that enhances the production of triacylglycerol (TAG), by genetictransformation of an oil-producing organism with said sequence in orderto be expressed in this organism, resulting in an active protein thatincreases the oil content of the organism. The nucleic acid sequence isderived from the sequence shown in SEQ ID NO. 1 from the Saccharomycescerevisiae YJR098c gene (genomic clone or cDNA) or from a nucleic acidsequence or cDNA that contains a nucleotide sequence coding for aprotein with an amino acid sequence that is 60% or more identical to theamino acid sequence as presented in SEQ ID No: 2.

The gene product, which we refer to as a TAG synthesis enhancing protein(TEP) is most likely not itself catalyzing the synthesis of TAG, but itspresence elevates the amount of TAG synthesized by other enzymes.

The instant invention pertains to a gene construct comprising a saidnucleotide sequence SEQ ID No: 1 of the instant invention, which isoperably linked to a heterologous nucleic acid.

The term operably linked means a serial organization e.g. of a promoter,coding sequence, terminator and/or further regulatory elements wherebyeach element can fulfill its original function during expression of thenucleotide sequence.

Further, a vector comprising the said nucleotide sequence SEQ ID No: 1of the instant invention is contemplated in the instant invention. Thisincludes also an expression vector which can harbor a selectable markergene and/or nucleotide sequences for the replication in a host celland/or the integration into the genome of the host cell.

Furthermore, this invention relates to a method for producing a TEP in ahost cell or progeny thereof including genetically engineered oil seeds,yeast and moulds or any other oil-accumulating organism, via theexpression of a construct in the cell. Of particular interest is theexpression of the nucleotide sequences of the present invention fromtranscription initiation regions that are preferentially expressed inplant seed tissues. It is further contemplated that an artificial genesequence encoding TEP may be synthesized, especially to provideplant-preferred codons. Cells containing a TEP as a result of theproduction of a TEP encoding sequence are also contemplated within thescope of the invention.

Further, the invention pertains a transgenic cell or organism containinga said nucleotide sequence and/or a said gene construct and/or a saidvector. The object of the instant invention is further a transgenic cellor organism which is an eucaryotic cell or organism. Preferably, thetransgenic cell or organism is a yeast cell or a plant cell or a plant.The instant invention further pertains said transgenic cell or organismhaving an increased biosynthetic pathway for the production ofsubstrates for the synthesis of triacylglycerol. A transgenic cell ororganism having increased oil content is also contemplated within thescope of this invention.

Further, the invention pertains a transgenic cell or organism whereinthe activity of TEP is increased in said cell or organism. The increasedactivity of TEP is characterized by an alteration in gene expression,catalytic activity and/or regulation of activity of the enzyme.Moreover, a transgenic cell or organism is included in the instantinvention, wherein the increased biosynthetic pathway for the productionof substrates for the production of triacylglycerol is characterizede.g. by the prevention of accumulation of undesirable fatty acids in themembrane lipids.

In a different embodiment, this invention also relates to methods ofusing a DNA sequence coding for a TEP for increasing the oil-contentwithin the cells of different organisms.

Further, the invention makes possible a process for elevating theproduction of triacylglycerol, which comprises growing transgenic cellsor organisms under conditions whereby the nucleotide sequence SEQ-ID No:1 is expressed in order to produce an protein in these cells with theability of enhancing the production of triacylglycerol.

Corresponding genes coding for TEP can be isolated from other organisms,especially yeast-type organisms, like e.g. Schizosaccharomyces pombe,Yarrowia lipolytica, Zygosaccharomyces rouxii, Saccharomyces cerevisiae,Emericella nidulans and Debaryomyces hansenii.

Transgenic organisms comprising, in their genome or on a plasmid, anucleic acid sequence SEQ ID No:1 according to the above, transferred byrecombinant DNA technology. One important type of transgenic organismcovered by this invention are commercially relevant plants in which saidnucleotide sequence preferably would be expressed under the control of astorage organ specific promoter. Alternatively, the nucleotide sequencecould also be expressed under the control of a seed-specific promoter orany other promoter suitable for tissue-specific high-level expression inplants.

A protein encoded by a DNA molecule according to SEQ ID NO. 1 or afunctional biologically active fragment thereof having TEP activity intransgenic organisms. Alternatively, the protein produced in anorganism, which has the amino acid sequence set forth in SEQ ID NO. 2 oran amino acid sequence with at least 60% homology to said amino acidsequence having TEP activity. Preferably the protein is isolated fromSaccharomyces cerevisiae.

Use of a protein according to SEQ ID No: 2 or derivatives of thatprotein having TEP activity for the increased production oftriacylglycerols.

Surprisingly, it has been found that the heterologous expression of theyeast TEP from Saccharomyces cerevisiae SEQ ID NO: 1 in Arabidopsisleads to a significantly increased triacylglyceride (storage oils)content in the seeds. The oil content was increased by approximately 5%,in one transgenic line even by 10%, compared with wild-type controlplants. The transgenic expression of the yeast TEP had no adverseeffects on the growth or other properties of the transformed plants.

The method according to the invention can be applied in principle to allplant species, in addition to the species Arabidopsis thaliana, which isemployed as model plant. The method according to the invention ispreferably applied to oil crops whose oil content is already naturallyhigh and/or for the industrial production of oils.

“Plant” organism or tissue, organ, part, cell or propagation materialthereof is generally understood as meaning any single- or multi-celledorganism or a cell, tissue, part or propagation material (such as seedsor fruit) of same which is capable of photosynthesis. Included for thepurpose of the invention are all genera and species of higher and lowerplants of the Plant Kingdom. Annual, perennial, monocotyledonous anddicotyledonous plants are preferred. Also included are mature plants,seeds, shoots and seedlings, and parts, propagation material (forexample tubors, seeds or fruits) and cultures derived from them, forexample cell cultures or callus cultures.

“Plant” encompasses all annual and perennial monocotyldedonous ordicotyledonous plants and includes by way of example, but not bylimitation, those of the genera Cucurbita, Rosa, Vitis, Juglans,Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna,Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium,Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis,Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio,Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium,Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea andPopulus.

Preferred plants are those from the following plant families:Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae,Compositae, Cruciferae, Cucurbitaceae, Labiatae, Leguminosae,Papilionoideae, Liliaceae, Linaceae, Malvaceae, Rosaceae, Rubiaceae,Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae,Tetragoniaceae, Theaceae, Umbelliferae.

Preferred monocotyledonous plants are selected in particular from themonocotyledonous crop plants such as, for example, the Gramineae family,such as rice, maize, wheat or other cereal species such as barley,millet and sorghum, rye, triticale or oats, and sugar cane, and allgrass species.

The invention is applied very particularly preferably to dicotyledonousplant organisms. Preferred dicotyledonous plants are selected inparticular from the dicotyledonous crop plants such as, for example,

-   -   Asteraceae such as Heliantus annuus (sunflower), tagetes or        calendula and others,    -   Compositae, especially the genus Lactuca, very particularly the        species sativa (lettuce) and others,    -   Cruciferae, particularly the genus Brassica, very particularly        the species napus (oilseed rape), campestris (beet), oleracea cv        Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and        oleracea cv Emperor (broccoli) and other cabbages; and the genus        Arabidopsis, very particularly the species thaliana, and cress        or canola and others,    -   Cucurbitaceae such as melon, pumpkin/squash or zucchini and        others,    -   Leguminosae, particularly the genus Glycine, very particularly        the species max (soybean), soya, and alfalfa, pea, beans or        peanut and others,    -   Rubiaceae, preferably the subclass Lamiidae such as, for example        Coffea arabica or Coffea liberica (coffee bush) and others,    -   Solanaceae, particularly the genus Lycopersicon, very        particularly the species esculentum (tomato), the genus Solanum,        very particularly the species tuberosum (potato) and melongena        (aubergine) and the genus Capsicum, very particularly the genus        annuum (pepper) and tobacco or paprika and others,    -   Sterculiaceae, preferably the subclass Dilleniidae such as, for        example, Theobroma cacao (cacao bush) and others,    -   Theaceae, preferably the subclass Dilleniidae such as, for        example, Camellia sinensis or Thea sinensis (tea shrub) and        others,    -   Umbelliferae, particularly the genus Daucus (very particularly        the species carota (carrot)) and Apium (very particularly the        species graveolens dulce (celeary)) and others;        and linseed, cotton, hemp, flax, cucumber, spinach, carrot,        sugar beet and the various tree, nut and grapevine species, in        particular banana and kiwi fruit.

Also encompassed are ornamental plants, useful or ornamental trees,flowers, cut flowers, shrubs or turf plants which may be mentioned byway of example but not by limitation are angiosperms, bryophytes suchas, for example, Hepaticae (liverworts) and Musci (mosses);pteridophytes such as ferns, horsetail and clubmosses; gymnosperms suchas conifers, cycades, ginkgo and Gnetatae; algae such as Chlorophyceae,Phaeophpyceae, Rhodophyceae, Myxophyceae, Xanthophyceae,Bacillariophyceae (diatoms) and Euglenophyceae. Plants within the scopeof the invention comprise by way of example and not by way oflimitation, the families of the Rosaceae such as rose, Ericaceae such asrhododendron and azalea, Euphorbiaceae such as poinsettias and croton,Caryophyllaceae such as pinks, Solanaceae such as petunias, Gesneriaceaesuch as African violet, Balsaminaceae such as touch-me-not, Orchidaceaesuch as orchids, Iridaceae such as gladioli, iris, freesia and crocus,Compositae such as marigold, Geraniaceae such as geranium, Liliaceaesuch as dracena, Moraceae such as ficus, Araceae such as cheeseplant andmany others.

Furthermore, plant organisms for the purposes of the invention arefurther organisms capable of being photosynthetically active such as,for example, algae, cyanobacteria and mosses. Preferred algae are greenalgae such as, for example, algae from the genus Haematococcus,Phaedactylum tricornatum, Volvox or Dunaliella. Synechocystis isparticularly preferred.

Most preferred are oil crops. Oil crops are understood as being plantswhose oil content is already naturally high and/or which can be used forthe industrial production of oils. These plants can have a high oilcontent and/or else a particular fatty acid composition which is ofinterest industrially. Preferred plants are those with a lipid contentof at least 1% by weight. Oil crops encompassed by way of example:Borvago officinalis (borage); Brassica species such as B. campestris, B.napus, B. rapa (mustard, oilseed rape or turnip rape); Cannabis sativa(hemp); Carthamus tinctorius (safflower); Cocos nucifera (coconut);Crambe abyssinica (crambe); Cuphea species (Cuphea species yield fattyacids of medium chain length, in particular for industrialapplications); Elaeis guinensis (African oil palm); Elaeis oleifera(American oil palm); Glycine max (soybean); Gossypium hirisutfum(American cotton); Gossypium barbadense (Egyptian cotton); Gossypiumherbaceum (Asian cotton); Helianthus annuus (sunflower); Linumusitatissimum (linseed or flax); Oenothera biennis (evening primrose);Olea europaea (olive); Oryza sativa (rice); Ricinus communis (castor);Sesamum indicum (sesame); Triticum species (wheat); Zea mays (maize),and various nut species such as, for example, walnut or almond.

“Total oil content” refers to the sum of all oils, preferably to the sumof the triacylglycerides.

“Oils” encompasses neutral and/or polar lipids and mixtures of these.Those mentioned in Table 1 may be mentioned by way of example, but notby limitation.

TABLE 1 Classes of plant lipids Neutrale lipids Triacylglycerol (TAG)Diacylglycerol (DAG) Monoacylglycerol (MAG) Polar lipidsMonogalactosyldiacylglycerol (MGDG) Digalactosyldiacylglycerol (DGDG)Phosphatidylglycerol (PG) Phosphatidylcholine (PC)Phosphatidylethanolamine (PE) Phosphatidylinositol (PI)Phosphatidylserine (PS) Sulfoquinovosyldiacylglycerol

Neutral lipids preferably refers to triacylglycerides. Both neutral andpolar lipids may comprise a wide range of various fatty acids. The fattyacids mentioned in Table 2 may be mentioned by way of example, but notby limitation.

TABLE 2 Overview over various fatty acids (selection) Nomenclature¹ Name16:0 Palmitic acid 16:1 Palmitoleic acid 16:3 Roughanic acid 18:0Stearic acid 18:1 Oleic acid 18:2 Linoleic acid 18:3 Linolenic acidγ-18:3-18:3 Gamma-linolenic acid* 20:0 Arachidic acid 22:6Docosahexaenoic acid (DHA)* 20:2 Eicosadienoic acid 20:4 Arachidonicacid (AA)* 20:5 Eicosapentaenoic acid (EPA)* 22:1 Erucic acid ¹Chainlength: number of double bonds *not naturally occurring in plants

Oils preferably relates to seed oils.

“Increase in” the total oil content refers to the increased oil contentin a plant or a part, tissue or organ thereof, preferably in the seedorgans of the plants. In this context, the oil content is at least 5%,preferably at least 10%, particularly preferably at least 15%, veryparticularly preferably at least 20%, most preferably at least 25%increased under otherwise identical conditions in comparison with astarting plant which has not been subjected to the method according tothe invention, but is otherwise unmodified. Conditions in this contextmeans all of the conditions which are relevant for germination, cultureor growth of the plant, such as soil conditions, climatic conditions,light conditions, fertilization, irrigation, plant protection treatmentand the like.

“Yeast TEP” generally refers to all those proteins which are capable ofincreasing the oil content in oil producing organisms, especiallymicroorganisms, yeast, fungi and plants and are identical to SEQ ID No:2 or have homology to SEQ ID No: 2.

Yeast refers to the group of unicellular fungi with a pronounced cellwall and formation of pseudomycelium (in contrast to molds). Theyreproduce vegetatively by budding and/or fission (Schizo-saccharomycesand Saccharomycodes, respectively).

Encompassed are what are known as false yeasts, preferably the familiesCryptococcaceae, Sporobolomycetaceae with the genera Cryptococcus,Torulopsis, Pityrosporum, Brettanomyces, Candida, Kloeckera,Trigonopsis, Trichosporon, Rhodotorula and Sporobolomyces and Bullera,and true yeasts (yeasts which also reproduce sexually; ascus),preferably the families endo- and saccharomycetaceae, with the generaSaccharomyces, Debaromyces, Lipomyces, Hansenula, Endomycopsis, Pichia,Hanseniaspora. Most preferred are the genera Saccharomyces cerevisiae,Pichia pastoris, Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Zygosaccharomyces rouxii, and Yarrowia lipolitica,Emericella nidulans, Aspergillus nidulans, Debaryomyces hansenii andTorulaspora hansenii.

Yeast TEP refers in particular to the polypeptide sequence SEQ ID No: 2.

Most preferably, yeast TEP refers to the yeast protein TEP as shown inSEQ ID NO: 2 and functional equivalents or else functionally equivalentportions of the above.

Functional equivalents refers in particular to natural or artificialmutations of the yeast protein TEP as shown in SEQ ID NO: 2 andhomologous polypeptides from other yeasts which have the same essentialcharacteristics of a yeast TEP as defined above. Mutations encompasssubstitutions, additions, deletions, inversions or insertions of one ormore amino acid residues.

The yeast TEP to be employed advantageously within the scope of thepresent invention can be found readily by database searches or byscreening gene or cDNA libraries using the yeast TEP sequence shown inSEQ ID NO: 2, which is given by way of example, or the nucleic acidsequence as shown in SEQ ID NO: 1, which encodes the latter, as searchsequence or probe.

Said functional equivalents preferably have at least 60%, particularlypreferably at least 70%, particularly preferably at least 80%, mostpreferably at least 90% homology with the protein of SEQ ID NO: 2.

Homology between two polypeptides is understood as meaning the identityof the amino acid sequence over the entire sequence length which iscalculated by comparison with the aid of the program algorithm GAP(Wisconsin Package Version 10.0, University of Wisconsin, GeneticsComputer Group (GCG), Madison, USA), setting the following parameters:

Gap Weight: 8 Length Weight: 2 Average Match: 2,912 Average Mismatch:−2,003

For example, a sequence with at least 80% homology with the sequence SEQID NO: 2 at the protein level is understood as meaning a sequence which,upon comparison with the sequence SEQ ID NO: 2 with the above programalgorithm and the above parameter set has at least 80% homology.

Functional equivalents also encompass those proteins which are encodedby nucleic acid sequences which have at least 60%, particularlypreferably at least 70%, particularly preferably at least 80%, mostpreferably at least 90% homology with the nucleic acid sequence with theSEQ ID NO: 1.

Homology between two nucleic acid sequences is understood as meaning theidentity of the two nucleic acid sequences over the entire sequencelength which is calculated by comparison with the aid of the programalgorithm GAP (Wisconsin Package Version 10.0, University of Wisconsin,Genetics Computer Group (GCG), Madison, USA), setting the followingparameters:

Gap Weight: 50 Length Weight: 3 Average Match: 10 Average Mismatch: 0

For example, a sequence which has at least 80% homology with thesequence SEQ ID NO: 1 at the nucleic acid level is understood as meaninga sequence which, upon comparison with the sequence SEQ ID NO: 1 withinthe above program algorithm with the above parameter set has a homologyof at least 80%.

Functional equivalents also encompass those proteins which are encodedby nucleic acid sequences which hybridize under standard conditions witha nucleic acid sequence described by SEQ ID NO: 1, the nucleic acidsequence which is complementary thereto or parts of the above and whichhave the essential characteristics for a yeast TEP.

“Standard hybridization conditions” is to be understood in the broadsense, but preferably refers to stringent hybridization conditions. Suchhybridization conditions are described, for example, by Sambrook J,Fritsch E F, Maniatis T et al., in Molecular Cloning (A LaboratoryManual), 2^(nd) edition, Cold Spring Harbor Laboratory Press, 1989,pages 9.31-9.57) or in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the conditionsduring the wash step can be selected from the range of high-stringencyconditions (with approximately 0.2×SSC at 50° C., preferably at 65° C.)(20×SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0). Denaturing agents suchas, for example, formamide or SDS may also be employed duringhybridization. In the presence of 50% formamide, hybridization ispreferably carried out at 42° C.

The invention furthermore relates to transgenic expression constructswhich can ensure a transgenic expression of a yeast TEP in a plantorganism or a tissue, organ, part, cells or propagation material of saidplant organism.

The definition given above applies to yeast TEP, with the transgenicexpression of a yeast TEP described by the sequence with the SEQ ID NO:2 being particularly preferred.

In said transgenic expression constructs, a nucleic acid moleculeencoding a yeast TEP is preferably in operable linkage with at least onegenetic control element (for example a promoter) which ensuresexpression in a plant organism or a tissue, organ, part, cell orpropagation material of same.

Especially preferred are transgenic expression cassettes wherein thenucleic acid sequence encoding a TEP is described by

-   a) a sequence with the SEQ ID NO: 1,-   b) a sequence derived from a sequence with the SEQ ID NO: 1 in    accordance with the degeneracy of the genetic code-   c) a sequence which has at least 60% identity with the sequence with    the SEQ ID NO: 1.

Operable linkage is understood as meaning, for example, the sequentialarrangement of a promoter with the nucleic acid sequence encoding ayeast TEP which is to be expressed (for example the sequence as shown inSEQ ID NO: 1 and, if appropriate, further regulatory elements such as,for example, a terminator in such a way that each of the regulatoryelements can fulfil its function when the nucleic acid sequence isexpressed recombinantly. Direct linkage in the chemical sense is notnecessarily required for this purpose. Genetic control sequences suchas, for example, enhancer sequences can also exert their function on thetarget/sequence from positions which are further removed or indeed fromother DNA molecules. Preferred arrangements are those in which thenucleic acid sequence to be expressed recombinantly is positioned behindthe sequence acting as promoter so that the two sequences are linkedcovalently to each other. The distance between the promoter sequence andthe nucleic acid sequence to be expressed recombinantly is preferablyless than 200 base pairs, particularly preferably less than 100 basepairs, very particularly preferably less than 50 base pairs.

Operable linkage and a transgenic expression cassette can both beeffected by means of conventional recombination and cloning techniquesas they are described, for example, in Maniatis T, Fritsch E F andSambrook J (1989) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor (NY), in Silhavy T J, Berman M Lund Enquist L W (1984) Experiments with Gene Fusions, Cold Spring HarborLaboratory, Cold Spring Harbor (NY), in Ausubel F M et al. (1987)Current Protocols in Molecular Biology, Greene Publishing Assoc. andWiley Interscience and in Gelvin et al. (1990) In: Plant MolecularBiology Manual. However, further sequences which, for example, act as alinker with specific cleavage sites for restriction enzymes, or of asignal peptide, may also be positioned between the two sequences. Also,the insertion of sequences may lead to the expression of fusionproteins. Preferably, the expression cassette composed of a promoterlinked to a nucleic acid sequence to be expressed can be in avector-integrated form and can be inserted into a plant genome, forexample by transformation.

However, a transgenic expression cassette is also understood as meaningthose constructs where the nucleic acid sequence encoding a yeast TEP isplaced behind an endogenous plant promoter in such a way that the latterbrings about the expression of the yeast TEP.

Promoters which are preferably introduced into the transgenic expressioncassettes are those which are operable in a plant organism or a tissue,organ, part, cell or propagation material of same. Promoters which areoperable in plant organisms is understood as meaning any promoter whichis capable of governing the expression of genes, in particular foreigngenes, in plants or plant parts, plant cells, plant tissues or plantcultures. In this context, expression may be, for example, constitutive,inducible or development-dependent.

The following are preferred:

a) Constitutive promoters

“Constitutive” promoters refers to those promoters which ensureexpression in a large number of, preferably all, tissues over asubstantial period of plant development, preferably at all times duringplant development (Benfey et al. (1989) EMBO J 8:2195-2202). A plantpromoter or promoter originating from a plant virus is especiallypreferably used. The promoter of the CaMV (cauliflower mosaic virus) 35Stranscript (Franck et al. (1980) Cell 21:285-294; Odell et al. (1985)Nature 313:810-812; Shewmaker et al. (1985) Virology 140:281-288;Gardner et al. (1986) Plant Mol Biol 6:221-228) or the 19S CaMV promoter(U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al. (1989) EMBO J8:2195-2202) are especially preferred. Another suitable constitutivepromoter is the Rubisco small subunit (SSU) promoter (U.S. Pat. No.4,962,028), the leguminB promoter (GenBank Acc. No. X03677), thepromoter of the nopalin synthase from Agrobacterium, the TR dualpromoter, the OCS (octopine synthase) promoter from Agrobacterium, theubiquitin promoter (Holtorf S et al. (1995) Plant Mol Biol 29:637-649),the ubiquitin 1 promoter (Christensen et al. (1992) Plant Mol Biol18:675-689; Bruce et al. (1989) Proc Natl Acad Sci USA 86:9692-9696),the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S.Pat. No. 5,683,439), the promoters of the vacuolar ATPase subunits, thepromoter of the Arabidopsis thaliana nitrilase-1 gene (GenBank Acc. No.:U38846, nucleotides 3862 to 5325 or else 5342) or the promoter of aproline-rich protein from wheat (WO 91/13991), and further promoters ofgenes whose constitutive expression in plants is known to the skilledworker. The CaMV 35S promoter and the Arabidopsis thaliana nitrilase-1promoter are particularly preferred.

b) Tissue-Specific Promoters

Furthermore preferred are promoters with specificities for seeds, suchas, for example, the phaseolin promoter (U.S. Pat. No. 5,504,200; BustosM M et al. (1989) Plant Cell 1(9):839-53), the promoter of the 2Salbumin gene (Joseffson L G et al. (1987) J Biol Chem 262:12196-12201),the legumine promoter (Shirsat A et al. (1989) Mol Gen Genet215(2):326-331), the USP (unknown seed protein) promoter (Bäumlein H etal. (1991) Mol Gen Genet 225(3):459-67), the napin gene promoter (U.S.Pat. No. 5,608,152; Stalberg K et al. (1996) L Planta 199:515-519), thepromoter of the sucrose binding proteins (WO 00/26388) or the legumin B4promoter (LeB4; Bäumlein H et al. (1991) Mol Gen Genet 225: 121-128;Bäumlein et al. (1992) Plant Journal 2(2):233-9; Fiedler U et al. (1995)Biotechnology (NY) 13(10):1090f), the Arabidopsis oleosin promoter (WO98/45461), and the Brassica Bce4 promoter (WO 91/13980).

Further suitable seed-specific promoters are those of the gene encodinghigh-molecular weight glutenin (HMG), gliadin, branching enyzme, ADPglucose pyrophosphatase (AGPase) or starch synthase. Promoters which arefurthermore preferred are those which permit a seed-specific expressionin monocots such as maize, barley, wheat, rye, rice and the like. Thepromoter of the lpt2 or lpt1 gene (WO 95/15389, WO 95/23230) or thepromoters described in WO 99/16890 (promoters of the hordein gene, theglutelin gene, the oryzin gene, the prolamin gene, the gliadin gene, theglutelin gene, the zein gene, the casirin gene or the secalin gene) canadvantageously be employed.

c) Chemically Inducible Promoters

The expression cassettes may also contain a chemically induciblepromoter (review article: Gatz et al. (1997) Annu Rev Plant PhysiolPlant Mol Biol 48:89-108), by means of which the expression of theexogenous gene in the plant can be controlled at a particular point intime. Such promoters such as, for example, the PRP1 promoter (Ward etal. (1993) Plant Mol Biol 22:361-366), a salicylic acid-induciblepromoter (WO 95/19443), a benzenesulfonamide-inducible promoter (EP 0388 186), a tetracyclin-inducible promoter (Gatz et al. (1992) Plant J2:397-404), an abscisic acid-inducible promoter EP 0 335 528) or anethanol-cyclohexanone-inducible promoter (WO 93/21334) can likewise beused. Also suitable is the promoter of the glutathione-S transferaseisoform II gene (GST-II-27), which can be activated by exogenouslyapplied safeners such as, for example, N,N-diallyl-2,2-dichloroacetamide(WO 93/01294) and which is operable in a large number of tissues of bothmonocots and dicots.

Particularly preferred are constitutive promoters, very particularlypreferred seed-specific promoters, in particular the napin promoter andthe USP promoter.

In addition, further promoters which make possible expression in furtherplant tissues or in other organisms such as, for example, E. colibacteria, may be linked operably with the nucleic acid sequence to beexpressed. Suitable plant promoters are, in principle, all of theabove-described promoters.

The nucleic acid sequences present in the transgenic expressioncassettes according to the invention or transgenic vectors can be linkedoperably with further genetic control sequences besides a promoter. Theterm genetic control sequences is to be understood in the broad senseand refers to all those sequences which have an effect on theestablishment or the function of the expression cassette according tothe invention. Genetic control sequences modify, for example,transcription and translation in prokaryotic or eukaryotic organisms.The transgenic expression cassettes according to the inventionpreferably encompass a plant-specific promoter 5′-upstream of thenucleic acid sequence to be expressed recombinantly in each case and, asadditional genetic control sequence, a terminator sequence3′-downstream, and, if appropriate, further customary regulatoryelements, in each case linked operably with the nucleic acid sequence tobe expressed recombinantly.

Genetic control sequences also encompass further promoters, promoterelements or minimal promoters capable of modifying theexpression-controlling properties. Thus, genetic control sequences can,for example, bring about tissue-specific expression which isadditionally dependent on certain stress factors. Such elements are, forexample, described for water stress, abscisic acid (Lam E and Chua N H,J Biol Chem 1991; 266(26): 17131-17135) and thermal stress (Schoffl F etal. (1989) Mol Gen Genetics 217(2-3):246-53).

Further advantageous control sequences are, for example, in theGram-positive promoters amy and SPO2, and in the yeast or fungalpromotors ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.

In principle all natural promoters with their regulatory sequences likethose mentioned above may be used for the method according to theinvention. In addition, synthetic promoters may also be usedadvantageously.

Genetic control sequences further also encompass the 5′-untranslatedregions, introns or nonencoding 3′-region of genes, such as, forexample, the actin-1 intron, or the Adh1-S intron 1, 2 and 6 (forgeneral reference, see: The Maize Handbook, Chapter 116, Freeling andWalbot, Eds., Springer, New York (1994)). It has been demonstrated thatthese may play a significant role in regulating gene expression. Thus,it has been demonstrated that 5′-untranslated sequences can enhance thetransient expression of heterologous genes. Translation enhancers whichmay be mentioned by way of example are the tobacco mosaic virus 5′leader sequence (Gallie et al. (1987) Nucl Acids Res 15:8693-8711) andthe like. They may furthermore promote tissue specificity (Rouster J etal. (1998) Plant J 15:435-440).

The transient expression cassette can advantageously contain one or moreof what are known as enhancer sequences in operable linkage with thepromoter, and these make possible an increased recombinant expression ofthe nucleic acid sequence. Additional advantageous sequences such asfurther regulatory elements or terminators may also be inserted at the3′ end of the nucleic acid sequences to be expressed recombinantly. Oneor more copies of the nucleic acid sequences to be expressedrecombinanly may be present in the gene construct.

Polyadenylation signals which are suitable as control sequences areplant polyadenylation signals, preferably those which correspondessentially to Agrobacterium tumefaciens T-DNA polyadenylation signals,in particular those of gene 3 of the T-DNA (octopine synthase) of the Tiplasmid pTiACHS (Gielen et al. (1984) EMBO J 3:835 et seq.) orfunctional equivalents thereof. Examples of particularly suitableterminator sequences are the OCS (octopin synthase) terminator and theNOS (nopaline synthase) terminator.

Control sequences are furthermore understood as those which makepossible homologous recombination or insertion into the genome of a hostorganism, or removal from the genome. In the case of homologousrecombination, for example, the coding sequence of the specificendogenous gene can be exchanged in a directed fashion for a sequenceencoding a dsRNA. Methods such as the cre/lox technology permit thetissue-specific, possibly inducible, removal of the expression cassettefrom the genome of the host organism (Sauer B (1998) Methods.14(4):381-92). Here, certain flanking sequences are added to the targetgene (lox sequences), and these make possible removal by means of crerecombinase at a later point in time.

A recombinant expression cassette and the recombinant vectors derivedfrom it may comprise further functional elements. The term functionalelement is to be understood in the broad sense and refers to all thoseelements which have an effect on generation, replication or function ofthe expression cassettes vectors or transgenic organisms according tothe invention. Examples which may be mentioned, but not by way oflimitation, are:

-   a) Selection markers which confer resistance to a metabolism    inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456),    antibiotics or biocides, preferably herbicides, such as, for    example, kanamycin, G 418, bleomycin, hygromycin, or phosph    nothricin and the like. Particularly preferred selection markers are    those which confer resistance to herbicides. The following may be    mentioned by way of example: DNA sequences which encode    phosphinothricin acetyltransferases (PAT) and which inactivate    glutamine synthase inhibitors (bar and pat gene),    5-enolpyruvylshikimate-3-phosphate synthase genes (EPSP synthase    genes), which confer resistance to Glyphosate    (N-(phosphonomethyl)glycine), the gox gene, which encodes    Glyphosate-degrading enzyme (Glyphosate oxidoreductase), the deh    gene (encoding a dehalogenase which inactivates dalapon),    sulfonylurea- and imidazolinone-inactivating acetolactate synthases,    and bxn genes which encode nitrilase enzymes which degrade    bromoxynil, the aasa gene, which confers resistance to the    antibiotic apectinomycin, the streptomycin phosphotransferase (SPT)    gene, which permits resistance to streptomycin, the neomycin    phosphotransferase (NPTII) gene, which confers resistance to    kanamycin or geneticidin, the hygromycin phosphotransferase (HPT)    gene, which confers resistance to hygromycin, the acetolactate    synthase gene (ALS), which confers resistance to sulfonylurea    herbicides (for example mutated ALS variants with, for example, the    S4 and/or Hra mutation).-   b) Reporter genes which encode readily quantifiable proteins and    which allow the transformation efficacy or the expression site or    time to be assessed via their color or enzyme activity. Very    particularly preferred in this context are reporter proteins    (Schenborn E, Groskreutz D. Mol Biotechnol. 1999; 13(1):29-44) such    as the “green fluorescent protein” (GFP) (Sheen et al. (1995) Plant    Journal 8(5):777-784), chloramphenicol transferase, a luciferase (Ow    et al. (1986) Science 234:856-859), the aequorin gene (Prasher et    al. (1985) Biochem Biophys Res Commun 126(3):1259-1268),    β-galactosidase, with β-glucuronidase being very particularly    preferred (Jefferson et al. (1987) EMBO J 6:3901-3907).-   c) Replication origins which allow replication of the expression    cassettes or vectors according to the invention in, for example, E.    coli. Examples which may be mentioned are ORI (origin of DNA    replication), the pBR322 ori or the P15A ori (Sambrook et al.:    Molecular Cloning. A Laboratory Manual, 2^(nd) ed. Cold Spring    Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).-   d) Elements which are required for agrobacterium-mediated plant    transformation such as, for example, the right or left border of the    T-DNA, or the vir region.

To select cells which have successfully undergone homologousrecombination or else cells which have succesfully been transformed, itis generally required additionally to introduce a selectable markerwhich confers resistance to a biocide (for example a herbicide), ametabolism inhibitor such as 2-deoxyglucose-6-phosphate (WO 98/45456) oran antibiotic to the cells which have successfully undergonerecombination. The selection marker permits the selection of thetransformed cells from untransformed cells (McCormick et al. (1986)Plant Cell Reports 5:81-84).

In addition, said recombinant expression cassette or vectors maycomprise further nucleic acid sequences which do not encode a yeast TEPand whose recombinant expression leads to a further increase in fattyacid biosynthesis. By way of example, but not by limitation, such aproOIL nucleic acid sequence which is additionally expressedrecombinantly can be selected from among nucleic acids encodingacetyl-CoA carboxylase (ACCase), glycerol-3-phosphate acyltransferase(GPAT), lysophosphatidate acyltransferase (LPAT), diacylglycerolacyltransferase (DAGAT) and phospholipid:diacylglycerol acyltransferase(PDAT). Such sequences are known to the skilled worker and are readilyaccessible from databases or suitable cDNA libraries of the respectiveplants.

An expression cassette according to the invention can advantageously beintroduced into an organism or cells, tissues, organs, parts or seedsthereof (preferably into plants or plant cells, tissues, organs, partsor seeds) by using vectors in which the recombinant expression cassettesare present. The invention therefore furthermore relates to saidrecombinant vectors which encompass a recombinant expression cassettefor a yeast TEP.

For example, vectors may be plasmids, cosmids, phages, viruses or elseagrobacteria. The expression cassette can be introduced into the vector(preferably a plasmid vector) via a suitable restriction cleavage site.The resulting vector is first introduced into E. coli. Correctlytransformed E. coli are selected, grown, and the recombinant vector isobtained with methods known to the skilled worker. Restriction analysisand sequencing may be used for verifying the cloning step. Preferredvectors are those which make possible stable integration of theexpression cassette into the host genome.

The invention furthermore relates to transgenic plant organisms ortissues, organs, parts, cells or propagation material thereof whichcomprise a yeast TEP as defined above, a transgenic expression cassettefor a yeast TEP or a transgenic vector encompassing such an expressioncassette.

Such a transgenic plant organism is generated, for example, by means oftransformation or transfection of the corresponding proteins or nucleicacids. The generation of a transformed organism (or a transformed cellor tissue) requires introducing the DNA in question (for example theexpression vector), RNA or protein into the host cell in question. Amultiplicity of methods is available for this procedure, which is termedtransformation (or transduction or transfection) (Keown et al. (1990)Methods in Enzymology 185:527-537). Thus, the DNA or RNA can beintroduced for example directly by microinjection or by bombardment withDNA-coated microparticles. The cell may also be permeabilizedchemically, for example with polyethylene glycol, so that the DNA mayreach the cell by diffusion. The DNA can also be introduced byprotoplast fusion with other DNA-comprising units such as minicells,cells, lysosomes or liposomes. Electroporation is a further suitablemethod for introducing DNA; here, the cells are permeabilized reversiblyby an electrical pulse. Soaking plant parts in DNA solutions, and pollenor pollen tube transformation, are also possible. Such methods have beendescribed (for example in Bilang et al. (1991) Gene 100:247-250; Scheidet al. (1991) Mol Gen Genet 228:104-112; Guerche et al. (1987) PlantScience 52:111-116; Neuhause et al. (1987) Theor Appl Genet 75:30-36;Klein et al. (1987) Nature 327:70-73; Howell et al. (1980) Science208:1265; Horsch et al. (1985) Science 227:1229-1231; DeBlock et al.(1989) Plant Physiology 91:694-701; Methods for Plant Molecular Biology(Weissbach and Weissbach, eds.) Academic Press Inc. (1988); and Methodsin Plant Molecular Biology (Schuler and Zielinski, eds.) Academic PressInc. (1989)).

In plants, the methods which have been described for transforming andregenerating plants from plant tissues or plant cells are exploited fortransient or stable transformation. Suitable methods are, in particular,protoplast transformation by polyethylene glycol-induced DNA uptake, thebiolistic method with the gene gun, what is known as the particlebombardment method, electroporation, the incubation of dry embryos inDNA-containing solution, and microinjection.

In addition to these “direct” transformation techniques, transformationmay also be effected by bacterial infection by means of Agrobacteriumtumefaciens or Agrobacterium rhizogenes and the transfer ofcorresponding recombinant Ti plasmids or Ri plasmids by infection withtransgenic plant viruses. Agrobacterium-mediated transformation is bestsuited to cells of dicotyledonous plants. The methods are described, forexample, in Horsch R B et al. (1985) Science 225: 1229f).

When agrobacteria are used, the expression cassette is to be integratedinto specific plasmids, either into a shuttle vector or into a binaryvector. If a Ti or Ri plasmid is to be used for the transformation, atleast the right border, but in most cases the right and left border, ofthe Ti or Ri plasmid T-DNA is linked to the expression cassette to beintroduced as flanking region.

Binary vectors are preferably used. Binary vectors are capable ofreplication both in E. coli and in Agrobacterium. As a rule, theycontain a selection marker gene and a linker or polylinker flanked bythe right and left T-DNA border sequence. They can be transformeddirectly into Agrobacterium (Holsters et al. (1978) Mol Gen Genet163:181-187). The selection marker gene, which is, for example, thenptII gene, which confers resistance to kanamycin, permits a selectionof transformed agrobacteria. The Agrobacterium which acts as hostorganism in this case should already contain a plasmid with the virregion. The latter is required for transferring the T-DNA to the plantcells. An Agrobacterium transformed in this way can be used fortransforming plant cells. The use of T-DNA for the transformation ofplant cells has been studied intensively and described (EP 120 516;Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam, Chapter V; An et al. (1985) EMBO J 4:277-287). Variousbinary vectors, some of which are commercially available, such as, forexample, pBI101.2 or pBIN19 (Clontech Laboratories, Inc. USA), areknown.

Further promoters which are suitable for expression in plants have beendescribed (Rogers et al. (1987) Meth in Enzymol 153:253-277; Schardl etal. (1987) Gene 61:1-11; Berger et al. (1989) Proc Natl Acad Sci USA86:8402-8406).

Direct transformation techniques are suitable for any organism and celltype. In cases where DNA or RNA are injected or electroporated intoplant cells, the plasmid used need not meet any particular requirements.Simple plasmids such as those from the pUC series may be used. If intactplants are to be regenerated from the transformed cells, it is necessaryfor an additional selectable marker gene to be present on the plasmid.

Stably transformed cells, i.e. those which contain the inserted DNAintegrated into the DNA of the host cell, can be selected fromuntransformed cells when a selectable marker is part of the insertedDNA. By way of example, any gene which is capable of conferringresistance to antibiotics or herbicides (such as kanamycin, G 418,bleomycin, hygromycin or phosphinothricin and the like) is capable ofacting as marker (see above). Transformed cells which express such amarker gene are capable of surviving in the presence of concentrationsof such an antibiotic or herbicide which kill an untransformed wildtype. Examples are mentioned above and preferably comprise the bar gene,which confers resistance to the herbicide phosphinothricin (Rathore K Set al. (1993) Plant Mol Biol 21(5):871-884), the nptII gene, whichconfers resistance to kanamycin, the hpt gene, which confers resistanceto hygromycin, or the EPSP gene, which confers resistance to theherbicide Glyphosate. The selection marker permits selection oftransformed cells from untransformed cells (McCormick et al. (1986)Plant Cell Reports 5:81-84). The plants obtained can be bred andhybridized in the customary manner. Two or more generations should begrown in order to ensure that the genomic integration is stable andhereditary.

The above-described methods are described, for example, in Jenes B etal. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, edited by SD Kung and R Wu, Academic Press,pp. 128-143, and in Potrykus (1991) Annu Rev Plant Physiol Plant MolecBiol 42:205-225). The construct to be expressed is preferably clonedinto a vector which is suitable for transforming Agrobacteriumtumefaciens, for example pBin19 (Bevan et al. (1984) Nucl Acids Res12:8711f).

Once a transformed plant cell has been generated, an intact plant can beobtained using methods known to the skilled worker. For example, calluscultures are used as starting material. The development of shoot androot can be induced in this as yet undifferentiated cell biomass in theknown fashion. The plantlets obtained can be planted out and used forbreeding.

The skilled worker is familiar with such methods for regenerating plantparts and intact plants from plant cells. Methods which can be used forthis purpose are, for example, those described by Fennell et al. (1992)Plant Cell Rep. 11: 567-570; Stoeger et al (1995) Plant Cell Rep.14:273-278; Jahne et al. (1994) Theor Appl Genet 89:525-533.

“Transgenic”, for example in the case of a yeast TEP, refers to anucleic acid sequence, an expression cassette or a vector comprisingsaid TEP nucleic acid sequence or to an organism transformed with saidnucleic acid sequence, expression cassette or vector or all thoseconstructs established by recombinant methods in which either

-   a) the nucleic acid sequence encoding a yeast TEP or-   b) a genetic control sequence, for example a promoter which is    functional in plant organisms, which is linked operably with said    nucleic acid sequence under a)-   c) (a) or (b)    are not in their natural genetic environment or have been modified    by recombinant methods, it being possible for the modification to    be, for example, a substitution, addition, deletion, inversion or    insertion of one or more nucleotide residues. Natural genetic    environment refers to the natural chromosomal locus in the source    organism or the presence in a genomic library. In the case of a    genomic library, the natural genetic environment of the nucleic acid    sequence is preferably retained, at least to some extent. The    environment flanks the nucleic acid sequence at least on one side    and has a sequence length of at least 50 bp, preferably at least 500    bp, particularly preferably at least 1000 bp, very particularly    preferably at least 5000 bp. A naturally occurring expression    cassette, for example the naturally occurring combination of the    promoter of a gene encoding for a yeast TEP with the corresponding    yeast TEP gene, becomes a transgenic expression cassette when the    latter is modified by non-natural, synthetic (“artificial”) methods    such as, for example, a mutagenization. Such methods are described    (U.S. Pat. No. 5,565,350; WO 00/15815; see also above).

Host or starting organisms which are preferred as transgenic organismsare, above all, plants in accordance with the above definition. Includedfor the purposes of the invention are all genera and species of higherand lower plants of the Plant

Kingdom, in particular plants which are used for obtaining oils, suchas, for example, oilseed rape, sunflower, sesame, safflower, olive tree,soya, maize, wheat and nut species. Furthermore included are the matureplants, seed, shoots and seedlings, and parts, propagation material andcultures, for example cell cultures, derived therefrom. Mature plantsrefers to plants at any desired developmental stage beyond the seedlingstage. Seedling refers to a young, immature plant at an earlydevelopmental stage.

The transgenic organisms can be generated with the above-describedmethods for the transformation or transfection of organisms.

The invention furthermore relates to the use of the transgenic organismsaccording to the invention and to the cells, cell cultures, parts—suchas, for example, in the case of transgenic plant organisms roots, leavesand the like—and transgenic propagation material such as seeds or fruitswhich are derived therefrom for the production of foodstuffs orfeedstuffs, pharmaceuticals or fine chemicals, in particular oils, fats,fatty acids or derivatives of these.

Besides influencing the oil content, the transgenic expression of ayeast TEP SEQ ID No: 1 or derivatives thereof in plants may mediate yetfurther advantageous effects such as, for example, an increased stressresistance. Such osmotic stress occurs for example in saline soils andwater and is an increasing problem in agriculture. Increased stresstolerance makes it possible, for example, to use areas in whichconventional arable plants are not capable of thriving for agriculturalusage.

The invention now having been generally described will be more readilyunderstood by reference to the following examples, which are includedfor the purpose of illustration only, and are not intended to limitscope of the present invention.

EXAMPLES

General Methods:

Unless otherwise specified, all chemicals were from Fluka (Buchs), Merck(Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sigma(Deisenhofen). Restriction enzymes, DNA-modifying enzymes and molecularbiological kits were from Amersham-Pharmacia (Freiburg), Biometra(Göttingen), Roche (Mannheim), New England Biolabs (Schwalbach), Novagen(Madison, Wis., USA), Perkin-Elmer (Weiterstadt), Qiagen (Hilden),Stratagen (Amsterdam, Netherlands), Invitrogen (Karlsruhe) and Ambion(Cambridgeshire, United Kingdom). The reagents used were employed inaccordance with the manufacturer's instructions.

For example, oligonucleotides can be synthesized chemically in the knownmanner using the phosphoamidite method (Voet, Voet, 2^(nd) edition,Wiley Press New York, pages 896-897). The cloning steps carried out forthe purposes of the present invention such as, for example, restrictioncleavages, agarose gel electrophoreses, purification of DNA fragments,transfer of nucleic acids to nitrocellulose and nylon membranes, linkingDNA fragments, transformation of E. coli cells, bacterial cultures,multiplication of phages and sequence analysis of recombinant DNA, arecarried out as decribed by Sambrook et al. (1989) Cold Spring HarborLaboratory Press; ISBN 0-87969-309-6. Recombinant DNA molecules weresequenced using an ABI laser fluorescence DNA sequencer following themethod of Sanger (Sanger et al. (1977) Proc Natl Acad Sci USA74:5463-5467).

Example 1

Reduction of Triacylglycerol Accumulation in Yeast Cells Lacking theYJR098c Gene

Yeast strains used in this study were congenic to the W303-1A (Thomas &Rothstein, 1989) background. An YJR098c mutant strain, H1223, with thegenotype MATα yjr098c::HIS3 ADE2 can 1-100 his3-11, 15 leu2-3, 112,trp1-1 ura3-1, was generated as described in Sandager et al., 2002. As awild type control, we used the strain SCY62 MATa ADE2 can 1-100his3-11,15 leu2-3, 112 trp1-1 ura3-1).

Yeast cells were cultivated at 30° C. on a rotary shaker in liquidsynthetic medium (Sherman et al., 1986) supplemented with 2% (wt/vol)glucose.

The lipid content of the yeast cells was determined as described byDahlqvist et al. (2000) and is presented as nmol of fatty acid (FA) permg dry weight yeast.

The lipid content of a mutant yeast strain H1223, in which the YJR098cgene was disrupted, was analyzed and compared to wild type yeast cells(strain SCY62). The lipid content was determined in yeast cellsharvested in stationary phase after 50 hours of cultivation in liquidsynthetic medium at 30° C. Lipids were extracted in chloroform,fractionated on TLC and quantified by GC analyses (Dahlqvist et al.,2000). The total lipid content, measured as nmol fatty acids (FA) perdry weight yeast, in the YJR098c mutant yeast was 18% less than in thewild type, see table 1. The main reason for this difference was alowered TAG content in the YJR098c mutant. Thus, the triacylglycerolamount in the mutant yeast was almost 36% lower than in the wild type,whereas the polar lipid content only differed slightly between theYJR098 mutant and the wild type yeast, see table 1.

In summary, this experiment shows that the product of the YJR098c genecontributes to TAG accumulation in yeast.

TABLE 1 Lipid content in yeast disrupted in the YJR098c gene. controlyeast YJR098c - mutant (nmol FA/mg) (nmol FA/mg) Sterol esters 28 25Triacylglycerol 180 116 Other neutral lipids 7 9 Polar lipids 95 104Total lipids 311 255

Example 2

Increased Accumulation of Triacylglycerol in Yeast Cells Expressing theYJR098c Gene in Combination with a Strong Promoter

For induced high level expression of the YJR098c gene, a 2439 bp DNAfragment, containing 29 bp up stream and 442 bp down stream of the gene,was amplified from wt W303 genomic DNA by using a 1:1 mixture of Taq andpfu DNA polymerases with the 5′ primer, CTTGTAGAGGTTAACTGGGGA, and the3′ primer, TGAATTGTCCTCGCTGTCAA. The resulting PCR product was blunt endcloned into the BamHI site of the GAL1 yeast expression plasmid pUS10,which is a selection marker variant of the GAL1 yeast expression plasmidpJN92 (Ronne et al., 1991) thus generating the plasmid pUS30. PUS 10 wasgenerated by removing the URA3 selection marker from the pJN92 plasmidby HindIII digestion and replacing it with the HIS3 gene, a 1768 bp DNAfragment that was blunt end cloned into the remaining part of theHINDIII digested pJN92. The wild type yeast strain SCY62 (MATa ADE2 can1-100 his3-11,15 leu2-3, 112 trp1-1 ura3-1), was transformed with thepUS30 and cultivated at 28° C. on a rotary shaker in synthetic medium(Sherman et al., 1986) lacking uracil and supplemented with 2% (vol/vol)glycerol and 2% (vol/vol) ethanol. The GAL1 promoter was induced after 6or 24 hours of growth by the addition of 2% (wt/vol) final concentrationof galactose. Cells were harvested after an additional 24 hours ofgrowth. Wild type cells SCY62 (MATa ADE2 can 1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1) transformed with the empty vector, pUS10, andcultivated under identical conditions were used as a control. The lipidcontent of the yeast cells was determined as described by Dahlqvist etal. (2000) and is presented as nmol of fatty acid (FA) per mg dry weightyeast.

The effect of high-level expression of the YJR098c gene on lipidaccumulation was studied by transforming the wild-type yeast strainSCY62 (Dahlqvist, et al., 2000) with a plasmid containing the YJR098cgene under control of the galactose-induced GALL promotor, see Table 2.High-level expression of the YJR098c gene from this promoter had nostrong effect on the growth rate as determined by optical densitymeasurements. The expression of the YJR098c gene was induced after 6 h(Table 2A) or 24 h (Table 2B) and cells were harvested after anadditional 24 hours of cultivation. The total lipid content, determinedas nmol fatty acids (FA) per mg yeast (Dahlqvist et al., 2000) in cellsexpressing the YJR098c gene from the GAL1 promoter was higher both at anearly (Table 2A) or late (Table 2B) stationary growth stage as comparedto cells transformed with an empty vector.

The elevated lipid content in cells expressing the YJR098c gene from theGAL1 promoter was entirely explained by an increased TAG content whereasthe content of polar lipids and sterol esters were unaffected.

In summary, the TAG content in yeast cells expressing YJR098c incombination with a strong promoter was increased with 26 to 28% ascompared to the control (Table 2A and 2 B), which demonstrates thepotential of the use of the YJR098c gene for increasing the oil contentin transgenic organisms including yeast.

TABLE 2 Lipid content in yeast that expresses the YJR098c gene incombination with the GAL1 promoter control yeast High level of YJR098c(nmol FA/mg) expression (nmol FA/mg) A Sterol esters 13 13Triacylglycerol 78 98 Other neutral lipids 9 9 Polar lipids 60 60 Totallipids 160 180 B Sterol esters 15 17 Triacylglycerol 142 182 Otherneutral lipids 9 11 Polar lipids 55 50 Total lipids 221 260

Example 3

Transgenic Plants Expressing YJR098c

For induced high level expression of the YJR098c gene in plants, a PCRfragment (2409 bp) was generated by the 5′ primer (CTT GTA GAG GTT AACTGG GGA) and the 3′primer (TGA ATT GTC CTC GCT GTC AA) adding 29 basesupstream of the gene and 442 bases downstream of the gene. The gene wascloned into the SmaI site of the vector pUC119 thus generating pUS 29.For Agrobacterium-mediated plant transformation a binary vector systemincluding the primary cloning vector pART7 with a CaMV35S promoter and abinary pART27 vector (Gleave A., 1992) were used. The pART7 vector witha napin promoter is a construct where the napin promoter fragment (1101bp) described by Stalberg (1993) replaced the CaMV35S promoter frompART7 only loosing the XhoI site of the polylinker in the process. TheYJR098c fragment were cut out from pUS 29 at the XbaI and SacI site andthen blunted into the pART7 vector with either the CaMV35S promoter,generating pEW 17 or with the napin promoter, generating pEW 14. Theentire cartridge including the promoter, the YJR098c gene and atranscriptional termination region were removed from the pART7 vector asa NotI fragment and introduced directly to the pART27 vector. Theplasmid was transformed into Agrobacterium tumefaciens.

Using floral dip essentially as described by Clough and Bent, 1998,plants of Arabidopsis thaliana were transformed with Agrobacteriumtumefaciens GV3101 harboring either of the plasmids pEWART27-14 andpEWART27-17. Entire plants (inflorescence and rosette) were submergedfor 20 to 30 sec in the infiltration media consisting of 5% sucrose and0.02% Silwet L-77 (Osi Specialties, Danbury, Conn.) plus resuspendedtransformed A. tumefaciens cells. Plants were then transferred to agrowth chamber with a photoperiod of 16 h of light at 21° C. and 8 h ofdark at 18° C. (70% humidity).

The seed oil content of T2 plants of the Arabidopsis transformants wasanalyzed by the use of conventional gas-liquid chromatography (GLC). Ascontrols, seeds from wild type plants were used. The level of expressionof the YJR098c gene in the seeds is determined by Northern blotanalysis.

The result of the measurements for the lines comprising the YJR098cconstruct showed a significantly higher total oil content in transgeniclines compared to the measurements of wild-type plants.

REFERENCES

-   Cases, S., Smith, S. J., Zheng, Y-W., Myers, H. M., Lear, S. R.,    Sande E., Novak, S., Collins, C., Welch, C. B., Lusis, A. J.,    Erickson, S. K., and Farese, R. V. (1998) Proc. Natl. Acad. Sci.,    USA 95, 13018-13023.-   Dahlqvist, A., Ståhl, U., Lenman, M., Banas, A., Lee, M., Sandager,    L., Ronne, H. and Stymne, S. (2000) Proc. Natl. Acad. Sci., USA 97,    6487-6492.-   Gleave, A. (1992) Plant Molecular Biology 20, 1203-1207.-   Lardizabal, K. D., Hawkins, D. J. and Thompson, G. A. (2001) DGAT2:    A New Diacylglycerol Acyltransferase Gene Family. JBC 276 (42)    38862-38869.-   Sandager, L., Gustavsson, M., Stahl, U., Dahlqvist, A., Wiberg, E.,    Banas, A., Lenman, M., Ronne, H., and Stymne, S. (2002) Storage    lipid synthesis is non-essential in yeast. Journal of Biological    Chemistry 277, 6478-6482-   Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Laboratory Course    Manual for Methods in Yeast Genetics, Cold Spring Harbor Lab. Press,    Plainview, N.Y.-   Stålberg, K., Ellerström, M., Josefsson, L.-G., and Rask, L. (1993)    Plant Molecular Biology 23, 671-683.-   Ronne, H., Carlberg, M., Hu, G.-Z. and Nehlin, J. O. (1991) Mol.    Cell. Biol. 11, 4876-5884.-   Thomas, B. J. and Rothstein, R. (1989) Cell 56, 619-630.

1. A method of increasing the total oil content in a plant or a tissue,organ, part, cell or propagation material thereof, comprising a)transgenically expressing in a plant or a tissue, organ, part, cell orpropagation material thereof a polypeptide, wherein the polypeptidecomprises the amino acid sequence as set forth in SEQ ID NO: 2; and b)selecting a transgenic plant, or a tissue, organ, part, cell orpropagation material thereof in which the total oil content in theplant, tissue, organ, part, cell or propagation material thereof isincreased as compared to the wild type.
 2. The method of claim 1,wherein the polypeptide is the polypeptide as set forth in SEQ ID NO: 2.3. The method of claim 1, wherein the plant is an oil crop.
 4. Themethod of claim 1, wherein the total oil content in a seed of thetransgenic plant is increased.
 5. A transgenic expression cassettecomprising a nucleic acid sequence under the control of a promoter whichis functional in a plant or a tissue, organ, part or cell thereof,wherein the nucleic acid sequence is selected from the group consistingof a) a nucleic acid sequence comprising the nucleic acid sequence asset forth in SEQ ID NO: 1; and b) a nucleic acid sequence encoding apolypeptide comprising the amino acid level-to sequence as set forth inSEQ ID NO: 2; and wherein expression of the nucleic acid sequenceresults in increased total oil content in the plant or the tissue,organ, part, cell or propagation material thereof.
 6. The transgenicexpression cassette of claim 5, wherein the nucleic acid sequence is thenucleic acid sequence as set forth in SEQ ID NO:
 1. 7. The transgenicexpression cassette of claim 5, wherein the nucleic acid sequence is anucleic acid sequence encoding the polypeptide as set forth in SEQ IDNO:
 2. 8. The tranagenic expression cassette of claim 5, wherein thepromoter is a seed-specific promoter.
 9. A transgenic vector comprisingthe expression cassette of claim
 5. 10. A transgenic plant or tissue,organ, part, cell or propagation material thereof, comprising theexpression cassette of claim
 5. 11. The transgenic plant of claim 10,wherein the plant is selected from the group consisting of Borvagoofficinalis, Brassica campestris, Brassica napus, Brassica rapa,Cannabis sativa, Cart hamus tinctorius, Cocos nucWera, Crainbeabyssinica, Cuphea species, Elaeis guinensis, Elaeis oleifera, Glycinemax, Gossypium hirsutum, Gossypium barbadense, Gossypium herbaceum,Helianthus annuus, Linum usitatissimum, Oenothera biennis, Oleaeuropaea, Oryza sativa, Ricinus communis, Sesamum indicum, Triticumspecies, Zea mays, walnut, and almond.
 12. A method for the productionof oils, fats or free fatty acids comprising extracting oils, fats orfree fatty acids from a transgenic plant or tissue, organ, part, cell orpropagation material thereof, wherein the transgenic plant or tissue,organ, part, cell or propagation material thereof is transformed with atransgenic expression cassette comprising a nucleic acid sequence underthe control of a promoter which is fractional in the plant or thetissue, organ, part or cell thereof, wherein the nucleic acid sequenceis selected from the group consisting of a) a nucleic acid sequencecomprising the nucleic acid sequence as set forth in SEQ ID NO: 1; andb) a nucleic acid sequence encoding a polypeptide comprising the aminoacid sequence as set forth in SEQ ID NO: 2; and wherein expression ofthe nucleic acid sequence results in increased total oil content in theplant or the tissue, organ, part, cell or propagation material thereofas compared to a wild type.
 13. The method of claim 12, wherein thenucleic acid sequence is the nucleic acid sequence as set forth in SEQID NO:
 1. 14. The method of claim 12, wherein the nucleic acid sequenceis a nucleic acid sequence encoding the polypeptide as set forth in SEQID NO:
 2. 15. A seed which is true breeding for an isolated nucleic acidmolecule encoding a polypeptide comprising the amino acid sequence asset forth in SEQ ID NO: 2 wherein expression of the polypeptide resultsin increased total oil content in the seed as compared to a wild typeseed.
 16. The seed of claim 15, wherein the polypeptide is thepolypeptide as set forth in SEQ ID NO:
 2. 17. A method of producing atransgenic plant having increased total oil content as compared to awild type variety of the plant, comprising the steps of: a) transforminga plant cell with an expression cassette comprising a nucleic acidsequence selected from the group consisting of i) a nucleic acidsequence comprising the nucleic acid sequence as set forth in SEQ ID NO:1; and ii) a nucleic acid sequence encoding a polypeptide comprising theamino acid sequence as set forth in SEQ ID NO: 2; b) generatingtransgenic plants from the plant cell; c) screening the trausgenicplants for increased total oil content; and d) selecting transgenicplants that demonstrate increased total oil content as compared to thewild type.
 18. The method of claim 17, wherein the nucleic acid sequenceis the nucleic acid sequence as set forth in SEQ ID NO:
 1. 19. Themethod of claim 17, wherein the nucleic acid sequence is a nucleic acidsequence encoding the polypeptide as set forth in SEQ ID NO: 2.